International Journal of Applied, Physical

and Bio-Chemistry Research (IJAPBCR)

ISSN(P): 2277-4793; ISSN(E): 2319-4448
Vol. 4, Issue 6, Dec 2014, 17-24
TJPRC Pvt. Ltd.

INHIBITION STUDY BY MOLECULAR DOCKING OF DIHYDROFOLATE REDUCTASE

OF ESCHERICHIA COLI WITH SOME CHALCONE MOLECULES
L. BENMEKHBI1, A. KRID2, L. BENCHARIF3 & M. BENCHARIF4
1,2,3,4

Laboratory of Molecular Chemistry, University of Constantine1, Constantine, Algeria

1

Department of Chemistry, University of Msila, Algeria

ABSTRACT
A srie of chalcone was synthesed by catalysed claisen schmit condensation and evalated for biological activity
against a various micro-organisme such Staphylococcus aures ATCC, Klebsiela pneumonia ATCC, Escherichia Coli
ATCC, Pseudomonas aeruginosa ATCC using the disk diffusion method and the minimum inhibitory concentration
(MIC). Molecular modeling (docking) studies show that 4,4'methyl mthoxychalcone represents the best inhibitor for the
E.coli dihydrofolate reductase (4DHFR). The crystals of the compound Ib were successfully grown by the solution growth
technique

at

room

temperature.

Single

crystal

XRD

studies

indicated

the

orthorhombic

structure

of

(E)-3-(2,6-Dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one.

KEYWORDS: Chalcones, Biological Activity, MIC, Dihydrofolate Reductase, X-Ray Diffraction

INTRODUCTION
For a structurally simple group of compounds, chalcones have displayed an impressive array for biological
activities1-5, among which anti-malarial6, anti protozoal anti-inflammatory7 immunomodulatory nitric oxid inhibition,
tyronase inhibition, cytotoxic8, and anticancer9, activities have been cited in literature10-13. These compounds obtained by
convenient synthesis methods strongly inhibit the polymerization of tubulin by binding to the colchicine -binding site the
relatively simple structure and high affinity of chalcones for the colchicines-binding site because of similarity of the tow
aryl group placements in the tow molecules has led to the synthesis and subsequent evaluation of a large number of
chalcones in our studies we interested in synthesis of such compounds and the evaluated of their anti-microbial activity14
and the correlation between the chemical structures of the studies compounds and their respective biological activities was
discussed15-16.
The biological activity of a compound can be characterised in its capacity to form weak bonds with the target
protein. The number and the type of these bonds play an important role in the forming and the stability of
protein-ligand complex16. In this context we are interested to study biological activity crystal structure and the molecular
docking interactions between these different molecules and the E.coli dihydrofolate reductase especially.

MATERIALS AND METHODS

Chemistry
Chalcones were synthesized by a base catalysed Claisen Shmith condensation reaction12 appropriately substuted
aldehyde and keton (scheme I). The method is attractive since it specifically generates the (E)-isomer. The IR spectra show
the caracteristique band for C=O at 1690-1695, C=C Ar at 1600-and 1475while the vinyl CH=CH appeared at 1295-1300
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L. Benmekhbi, A. Krid, L. Bencharif & M. Bencharif

Cm-1. From HNMR spectra all chalcones were geometrically pure and with Trans configuration JHa-HB=15.50, 15.60Hz.
Saturation of the double bond or variation of the aliphatic part results in loss of the anti-inflammatory activity.
Microbiology
Derivatives 1a-1e was tested for in vitro anti-microbial activity against:
Staphylococcus aureus ATCC, Klebsiela pneumonia ATCC, Escherichia Coli ATCC Pseudomonas aeruginosa
ATCC. Using the:
The diffusion method and the minimum inhibitory concentration (MIC)
The Diffusion Method
Each disk contain 200mg of the test compound for this method Muller Hinton agar was melted at 100C and after
cooling to 56 C was poured into Petri plates of 9cm diameter in quantities of 18 ml, left on the flat surface to solidify and
the surface of the medium was dried at 37C, then the culture of each bacteria and yeast strain after being kept in
Mueller Hinton broth to 10-5 cfu ml-1 were pipetted into the Mueller Hinton agar plate prepared as described above, the
surface of the medium was allowed to dry . The 10 mg ml-1 in DMSO compound impregnated discs were applied to the
surface of incubated plates. The Petri plates were placed in an incubator at 37C after 18h of incubation the Petri plates
were examined and it was found that all the test compounds exhibited different degrees og antibacterial activity or
inhibitory action.
The Minimum Inhibitory Concentration (MIC)
The MIC of these compounds was determined by the micro broth dilution technique using Muller-Hinton Broth.
Serial tow fold dilution ranged from 2500 to 2.4 mg-1 for compounds.
The inoculum was prepared in broth which had been kept overnight at 37C and which had been diluted with
Muller Hinton Broth to give a final concentration of 10-5 mg.ml-1 in the test tray. The trays were covered and placed in
plastique bags to prevent drying after incubation at 37C for 18-24. The MIC was defined as the lowest concentration of
compound giving complete inhibition of visible growth.
The values of the antimicrobial activity of the compounds are given in table 1 and 2.
Table 1: Antimicrobial Activity of the Synthesized Chalcone I (a-e)
Using the Diffusion Mthode, (Inhibition of Zone Diameter mm)
Compound
Ia
Ib
Ic
Id
Ie

Escherichia
coli
20
18
16
14
24

Pseudomonas
aeruginosa
18
20
16
14
-

Klebsiella
pneumoniae
14
16
20
12
12

Proteus
mirabilis
20
16
14
-14

Table 2: Results of MIC of the Synthesized Chalcone I (a-e)

Compd No
Ia
Ib
Impact Factor (JCC): 1.1514

Escherichia
coli
24
56

Pseudomonas
aeruginosa
125
56

Klebsiella
pneumoniae
256
120

Proteus
mirabilis
56
120
Index Copernicus Value (ICV): 3.0

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Inhibition Study by Molecular Docking of Dihydrofolate Reductase of Escherichia coli with Some Chalcone Molecules

Ic
Id
Ie

125
412
1.25

250
420
-

56
420
420

456
-420

DISCUSSIONS
All the tested compounds exihibited different degrees of antimicrobial activities or inhibitory actions. The most
susceptible organisms were the Escherichia coli and Pseudomonas aeruginosa followed by Klebsiella pneumoniae and the
lowest inhibitory effect was encountered in the case of Proteus mirabilis.
The highest degree of inhibition were recorded for the compounds Ia and Ie with Escherichia coli followed by
Ib and Ic with Pseudomonas aeruginosa and Klebsiella pneumoniae, while the lowest degree of inhibition was recrded
for the Id with all the different bacterial species.

RESULTS AND DISCUSSIONS

General Procedure of the Preparation of Chalcones
To a solution of 1.96g 4-mthoxy actophenone (10mmol) in ethanol an aqueous solution of 10cm3 10/ NaOH.
The resulting solution was heated to 80C and substituted benzaldhydes (10mmol) were added with constant stiring. The
reaction mixture was kept stirring at this T for 3-4h cooled to room temperature and was allowed to stand overnight
(scheme 1) the solid product separated was collected by filtration dried and recrystallized from ethanol to give I(a-e) in
75-90/ yields.

O
O
R

benzaldhyde

O
3HC

R'

C2H5OH
NaOH/H2O

actophnone

R'
chalcone

Scheme 1
Ia

R=H

R = H

chalcone

Ib

R = 2,6 Cl

R =OCH3

2,6, 4' dichloro methoxychalcone

Ic

R=H

R = OCH3

4 -mthoxychalcone

Id

R = OCH3

R = OCH3

4,4-dimthoxychalcone

Ie

R = Me

R' = OCH3

4,4'methyl mthoxychalcone

Crystal Growth and Characterization of (E)-3-(2,6-Dichlorophenyl)-1-(4-methoxyphenyl) prop-2-en-1-one (Ib)

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L. Benmekhbi, A. Krid, L. Bencharif & M. Bencharif

The second compound Ib (E)-3-(2,6-Dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one

was filtered and

purified and recrystallization in ethanol17. Crystal suitable for x-ray analysis was grown by slow evaporation of an acetone
solution at room temperature.
The molecular structure of (Ib), and the atomic numbering used, is illustrated in (Figure 1). A diagram of the
layered crystal packing in the unit cell of (Ib) is shown in Figure 2. A substituted chalcone adopts an E configuration with
respect to the C=C bond of the enone unit. The molecule is not planar, as can be seen from the dihedral angle of 6.21 (7)
between the two rings. The Crystal structure can be described by two types of crossed layers, parallel to (110) and (110)
respectively (Figure 2). The packing is stabilized by Van der Walls interactions and by CH interactions resulting in
the formation of three dimensional network (Table 1).

Figure 1: The Structure of the Title Compound with the Atomic Labeling Scheme
Displacements are drawn at the 50% probability level.

Figure 2: A Diagram of the Layered Crystal Packing in (I), Viewed Down the c Axis
Geometries of intermolecular interactions obtained from structural analysis of the compound Ib are in the Table 3
Table 3: Hydrogen-Bond Geometry (A, _)
DH_ _ _A
C4H4_ _ _Cg1i
C7H7_ _ _Cg2i

Impact Factor (JCC): 1.1514

DH
0.95
0.95

H_ _ _A
2.84
2.85

D_ _ _A
3.727
3.360

DH_ _ _A
157
115

Index Copernicus Value (ICV): 3.0

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Inhibition Study by Molecular Docking of Dihydrofolate Reductase of Escherichia coli with Some Chalcone Molecules

The Crystal Data and Structure Refinement for the Compound

Crystal Data
C16H12Cl2O2

F000 = 632

Mr = 307.16

Dx = 1.445 Mg m3

Orthorhombic, P212121

Mo K radiation
= 0.71073

Hall Symbol: P 2ac 2ab Reflections

Cell Parameters from 3041

a = 6.4793 (2)

= 2.427.4

b = 12.9807 (5)

= 0.46 mm1

c = 16.7819 (8)

T = 100 K

V = 1411.46 (10) 3

Prism, colourless

Z = 4 0.37 0.28 0.2 mm

0.37 0.28 0.2 mm

Data Collection
Bruker APEXII Diffractometer

2964 Reflections with I > 2(I)

Monochromator: graphite

Rint = 0.029

T = 100 K

max = 27.4

CCD rotation images, thin slices scans

min = 3.5

Absorption correction: multi-scan

(SADABS, Bruker, 1998)

h = 68

Tmin = 0.824, Tmax = 0.913

k = 1516

6643 measured reflections

l = 2021

3211 independent reflections

Refinement
Refinement on F2

Hydrogen Site Location: Inferred from Neighbouring Sites

Least-squares matrix: full

H-atom parameters constrained

R[F2 > 2(F2)] = 0.036

w = 1/[2(Fo2) + (0.0409P)2 + 0.5074P]

where P = (Fo2 + 2Fc2)/3

wR(F2) = 0.090

(/) max = 0.002

S = 1.05

max = 0.51 e 3

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22

L. Benmekhbi, A. Krid, L. Bencharif & M. Bencharif

3211 reflections

min = 0.20 e 3

182 parameters

Extinction correction: none

Theoretical Studies
Calculations carried out with the code Arguslab describe the potentialities of the active site to estabilish various
bonds with the ligand notably in the interactions of enzyme-inhibitor type.
The energies of interactions of these molecules with dihydrofolate rductase (4DHFR) are represented in table 4:
Table 4: The Energies of Interactions of these Molecules with 4DHFR
Compound
No
Ia
Ib
Ic
Id
Ie

Inhibition Zone
(mm)
20
18
16
14
24

The MIC
(g/l)
24
56
125
412
1.25

G
Kcal/Mol
-10,9227
-10,6451
-10,4504
-10,2815
-11,0496

DISCUSSIONS
By analysing the energies of interactions it is shown that compound Ie form the best complex with the lowest
energy. The main interactions found in this complexe are tow hydrogen bounds at 2.907129 A and 2.9994 A with Ala29
and Arg52 amino acids respectively. The simulation demonstrates that a hydrophobic interaction is formed between
C 4and C of leu28 at a distance of 2.0490061 A. it is important to note that the stability of the complex is
characterised also by - interaction between the ring A of the Ie and the benzenic ring of Phe31 amino acid. (Figure 3)

Figure 3: Complex Shown the Interactions Found between the Ligand and the Dihydrofolate Reductase (4DHFR)

CONCLUSIONS
In the first part of this work we were interesting on the biological activity of 5 different substituted chalcones by
the evaluation zone of inhibition and the MIC. Crystal of the compound was successfully grown by the solution growth
technique at room temperature. The various functional groups present in the compound were identified using FTIR
spectrum. Single crystal XRD studies indicated the orthorhombic structure of the crystal.
Impact Factor (JCC): 1.1514

Index Copernicus Value (ICV): 3.0

23

Inhibition Study by Molecular Docking of Dihydrofolate Reductase of Escherichia coli with Some Chalcone Molecules

The second part consists to study the inhibition of E.coli 4DHFR by Molecular Docking simulations. We found
that the best inhibition was found with Ie followed by Ia. The others have also different inhibitions. It is very important to
note that there is a good correlation between the the biological activities evaluated (inhibition zone and MIC) and the
interactions energies calculated with Docking technique.

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L. Benmekhbi, A. Krid, L. Bencharif & M. Bencharif

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Impact Factor (JCC): 1.1514

Index Copernicus Value (ICV): 3.0